Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

Background

Target enrichment and resequencing is a widely used approach for identification of cancer genes and genetic variants associated with diseases. Although cost effective compared to whole genome sequencing, analysis of many samples constitutes a significant cost, which could be reduced by pooling samples before capture. Another limitation to the number of cancer samples that can be analyzed is often the amount of available tumor DNA. We evaluated the performance of whole genome amplified DNA and the power to detect subclonal somatic single nucleotide variants in non-indexed pools of cancer samples using the HaloPlex technology for target enrichment and next generation sequencing.

Results

We captured a set of 1528 putative somatic single nucleotide variants and germline SNPs, which were identified by whole genome sequencing, with the HaloPlex technology and sequenced to a depth of 792–1752. We found that the allele fractions of the analyzed variants are well preserved during whole genome amplification and that capture specificity or variant calling is not affected. We detected a large majority of the known single nucleotide variants present uniquely in one sample with allele fractions as low as 0.1 in non-indexed pools of up to ten samples. We also identified and experimentally validated six novel variants in the samples included in the pools.

Conclusion

Our work demonstrates that whole genome amplified DNA can be used for target enrichment equally well as genomic DNA and that accurate variant detection is possible in non-indexed pools of cancer samples. These findings show that analysis of a large number of samples is feasible at low cost, even when only small amounts of DNA is available, and thereby significantly increases the chances of indentifying recurrent mutations in cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-856) contains supplementary material, which is available to authorized users.     

Medicinal plants resources of Western Himalayan Palas Valley,Indus Kohistan,Pakistan: Their uses and degrees of risk of extinction

Present study was intended with the aim to document the pre-existence traditional knowledge and ethnomedicinal uses of plant species in the Palas valley. Data were collected during 2015–2016 to explore plants resource, their utilization and documentation of the indigenous knowledge. The current study reported a total of 65 medicinal plant species of 57 genera belonging to 40 families. Among 65 species, the leading parts were leaves (15) followed by fruits (12), stem (6) and berries (1), medicinally significant while, 13 plant species are medicinally important for rhizome, 4 for root, 4 for seed, 4 for bark and 1 each for resin. Similarly, thirteen species were used as a whole while twelve species as partial for medicinal purpose. Further, it is concluded that every part of plants such as bulb, rhizome, roots, barks, leaves, flowers, fruit and seed were used for various ailments. Moreover, among 65 plants species, 09 species are threatened and placed into Endangered (EN) and Least Concern (LC) categories of IUCN. The recorded data are very useful and reflects the significance of the Palas valley as medicinal plants resource area.     

Biochar application to rice with 15N-labelled fertilizers,enhanced leaf nitrogen concentration and assimilation by improving morpho-physiological traits and soil quality

Leaf nitrogen (N) concentration plays an important role in biochemical and physiological functions, and N availability directly influences rice yield. However, excessive N fertilization is considered to be a root cause of environmental issues and low nitrogen use efficiency. Therefore, the selection of appropriate nutrient management practices and organic amendments is key to maximizing nitrogen uptake and maintaining high and sustainable rice production. Here, we evaluated the effects of different ¹⁵N-labelled nitrogen sources (urea, ammonium nitrate, and ammonium sulfate at 315 kg ha⁻¹) with or without biochar (30 t ha⁻¹) on paddy soil properties, root growth, leaf gas exchange, N metabolism enzymes, and N uptake in the early and late seasons of 2019. We found significant differences among N fertilizer sources applied with or without biochar (P < 0.05). Across the seasons, the combination of biochar with N fertilizers significantly increased soil organic carbon by 51.21% and nitrogen availability by 27.51% compared with N fertilizers alone. Correlation analysis showed that rice root morphological traits were strongly related to soil chemical properties, and higher root growth was measured in the biochar treatments. Similarly, net leaf photosynthetic rate averaged 9.34% higher, chlorophyll (Chl) a concentration 12.91% higher, and Chl b concentration 10.05% higher in the biochar treatments than in the biochar-free treatments across the seasons. Notably, leaf ¹⁵N concentration was 23.19% higher in the biochar treatments in both seasons. These results illustrated higher activities of N metabolism enzymes such as NR, GS, and GOGAT by an average 23.44%, 11.26% and 18.16% in the biochar treatments across the seasons, respectively. The addition of biochar with synthetic N fertilizers is an ecological nutrient management strategy that can increase N uptake and assimilation by ameliorating soil properties and improving the morpho-physiological factors of rice.     

Multivariate analysis for various quantitative traits in wheat advanced lines

Genetic diversity play key role in the germplasm improvement which is directly correlated with the crop production. Various statistical techniques have been used to study diversity among different genotypes. Among these techniques multivariate is most frequently used one for the genetic association of genotypes. In the present study a total of 64 advance lines included one check cultivar were evaluated under the field conditions of Cereal Crop Research Institute, Pirsabaq Nowshera, Pakistan during September 2017. Data were recorded for nine different parameters. Multivariate analysis divided the total 64 genotypes into four groups. The first five PCs with Eigen values > 1 contributed 86.95% of the variability amongst genotypes. Characters with maximum values in PC1 were Spikelets spike^-1 (SPPS) (0.732), spike length (SPL) (0.722) and biological yield (BY) (0.607), PC2 comprised of 100-grain weight (TGW) (0.605), grain yield (GY) (0.482) while days to heading (DH) (0.393), for PC3 major contributors were BY (0.550) and number of tillers meter square^-1 (NTPS) (0.289), the contribution of PC4 were flag leaf area (FLA) (0.716) and SPL (0.298) and the maximum values for various traits in PC5 were SPPS (0.732), SPL (0.722) and BY (0.607). From the findings of present study best performing lines can be directly recommended for general cultivation or to be used in future breeding programs.     

Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression

International Journal of Peptide Research and Therapeutics - Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent...     

Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil

The present experiment was designed to isolate bacterial strains from the brick kiln soil and to check the activity and enzyme kinetics of amylase from these isolates. The bacterial colonies were isolated from soil samples through the serial dilution method. The bacterial isolates were identified through morphological, electron microscopic and molecular analysis. The 16S ribosomal RNA sequences of the isolates IR-1, IR-2, IR-3, IR-8, and IR-9 showed high similarities with Bacillus tequilensis, Bacillus paramycoides, Proteus alimentorum, Bacillus wiedmannii, and Pseudomonas aeruginosa, respectively. All of the bacterial isolates showed a positive catalase activity except IR-9. Furthermore, the isolates showed variable antagonistic effects against different bacterial pathogens. All of the strains produced indole acetic acid (IAA), and the concentrations increased in the presence of tryptophan application. The isolates showed the amylase enzyme activity and maximum activity of isolates was achieved in 4% starch concentration. The IR-9 isolate showed the highest amylase activity of 5.9 U/ml. The V_max values of the extracellular amylase from different bacterial isolates ranged between 12.90 and 50.00 IU ml⁻¹. The lowest K_m value of 6.33 mg starch was recorded for IR-8 and the maximum K_cat value of 2.50 min⁻¹ was observed for IR-3. The amylase activity of the isolates was significantly affected by a range of different incubation time, temperature, and pH values. Further tests are required before the potential utilization of these isolates for amylase production, and in the biopesticide and biofertilizer applications.     

Peristaltic motion of Phan–Thien–Tanner fluid in the presence of slip condition

This investigation considers the peristaltic flow of a Phan–Thien–Tanner fluid in the presence of slip condition and induced magnetic field. By use of the long wavelength and low Reynolds number approximations, closed form series solutions for stream function, pressure gradient, magnetic force function, axial induced magnetic field, and current density were obtained. The pressure gradient and frictional forces per wavelength were computed by numerical integration. The velocity slip condition in terms of shear stress is taken into account. Graphical results show the comparison between no-slip and viscous fluid cases. Pumping and trapping phenomena are discussed.     

Endoplasmic reticulum remodeling tunes IP₃-dependent Ca²+ release sensitivity

The activation of vertebrate development at fertilization relies on IP₃-dependent Ca²⁺ release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP₃ receptors within ER patches have a higher sensitivity to IP₃ than those in the neighboring reticular ER. The lateral diffusion rate of IP₃ receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP₃ receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP₃ receptors through ER remodeling is sufficient to sensitize IP₃-dependent Ca²⁺ release. Mathematical modeling supports this concept of ‘geometric sensitization’ of IP₃ receptors as a population, and argues that it depends on enhanced Ca²⁺-dependent cooperativity at sub-threshold IP₃ concentrations. This represents a novel mechanism of tuning the sensitivity of IP₃ receptors through ER remodeling during meiosis.     

Nicotinamide inhibits alkylating agent-induced apoptotic neurodegeneration in the developing rat brain

Background

Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain.

Methodology/Principal Findings

Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment.

Conclusion

Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.     

Comparison of Phenacoccus solenopsis specimens from different regions of Pakistan using COI molecular barcoding (Hemiptera: Pseudococcidae)

Because correct identification of insects is crucial for pest management involving chemical or biological control agents, we have used a molecular approach to identify and characterize specimens of the cotton pest Phenacoccus solenopsis Tinsley (Sternorrhyncha: Pseudococcidae) present in different regions of Pakistan. The specimens were analyzed through DNA sequence analysis of their mitochondrial COI (mtCOI) gene using an improved procedure that could distinguish between the pest and its associated parasitoid. Our analysis showed no variation among the mealybug specimens from different geographical locations of Pakistan and confirmed that this is the same species and haplotype that is infesting cotton plants in other parts of Asia. This information will assist in the development of biological control programs against P. solenopsis in Pakistan and other Asian countries.